Even though the analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity, critical mechanisms by which it produces toxicity within the cell are unknown. The prevailing hypothesis is covalent binding of a toxic metabolite to crucial proteins and subsequent inhibition of vital cellular functions is responsible for the cytotoxicity. Even though it has been known for a number of years that covalent binding is via acetaminophen bound to cysteine groups on proteins, only recently have tools been available to identify the specific proteins to which acetaminophen covalently binds. In recent work we developed immunological assays which are specific for acetaminophen covalently bound to protein. These assays were utilized to determine the relationship between acetaminophen hepatotoxicity and covalent binding to subcellular fractions, individual hepatocytes, and specific proteins. In Western immunoblot studies it was shown that the principal liver protein to which acetaminophen covalently bound was a 55 kDa cytosolic protein. In preliminary data generated for this proposal the 55 kDa protein was isolated and 85 amino acids were sequenced from 7 internal peptides. Comparison of the sequence using computer data bases indicated that the protein had a 97% homology with the deduced amino acid sequence from a cDNA clone of a 56 kDa Selenium Binding Protein. It has been hypothesized to be a cellular regulatory protein. In this proposal other proteins to which acetaminophen covalently binds will be isolated and characterized by various methods including amino acid sequence analysis. The importance of covalent binding to specific proteins will be determined by comparing acetaminophen binding to a nontoxic analog which also binds to protein. In addition, it will be determined if other model hepatotoxins covalently bind to this Selenium Binding Protein.